Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for theses references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The primary cells of the immune system are the small white blood cells, called lymphocytes, derived from stem cells in the bone marrow. The differentiation of one class of lymphocytes is completed in the thymus gland. Accordingly, these lymphocytes are called T cells.
T cells circulate through the blood and lymphatic vessels of the body and are able to detect and react against foreign invaders, allergens, tumors and auto antigens. Despite their uniform morphology observed under the microscope, T cells consist of a heterogeneous population of three major subsets: the cytotoxic cells that destroy virus-infected cells; and two subsets, designated helper and suppressor cells, which regulate the antibody producing B cells.
A T cell clone is a T cell from which a population of identical T cells is derived by clonal expansion.
The molecular nature of the T cell antigen receptor was reported in the early 1980's by several research groups (1, 2, 3). As shown in these reports the T cell antigen receptor is a heterodimeric glycoprotein composed of two glycosylated polypeptides, one of which is designated the alpha chain and the other of which is designated the beta chain. A T cell antigen receptor is normally present on the surface of each T cell. Each T cell recognizes a single antigenic determinant in association with a protein of a major histocompatibility complex (MHC protein). All T cells derived from a T cell clone contain identical T cell antigen receptors and recognize the same antigenic determinant in association with the same MHC protein.
The alpha and beta chains of the T cell antigen receptor of a T cell clone are each composed of a unique combination of domains designated variable (V), diversity (D), joining (J) and constant (C) (4, 5). In each T cell clone the combination of V, D and J domains of both the alpha and the beta chains participates in antigen recognition in a manner which is uniquely characteristic of that T cell clone and defines a unique binding site, also known as the idiotype of the T cell clone. In contrast the C domain does not participate in antigen binding.
In addition to the alpha and beta chains, a third distinct gene, designated T cell receptor gamma gene, has been isolated. The sequence and organization of the gamma gene are similar to immunoglobulin genes and genes for the alpha and beta chain of the T cell antigen receptor (6). The gamma gene is expressed only in T cells. Tonegawa and coworkers suggest that T cell antigen receptors of some T cells are made up of a gamma chain and a beta chain (7).
The alpha and beta chains of the T cell antigen receptor of a T cell clone also define a plurality of antigenic determinants which can be recognized by antibodies directed to the antigen receptor. An antibody which reacts solely with the T cell clone or inhibits antigen binding only to the antigen receptor of the T cell clone against which it is raised is defined to be anti-idiotypic (2, 8, 9). If an antigenic determinant defined by the alpha or beta chains, or both, is present on the surface of, or associated with, a relatively limited number of T cell clones, the determinant is designated a minor framework determinant of the T cell antigen receptor(9, 10). If an antigenic determinant defined by the alpha or beta chain, or both, is present on the surface of, or associated with, a relatively large number or all T cell clones, the determinant is designated a major framework determinant of the T cell antigen receptor (11).
Although the demarcation between being present on a relatively limited number of T cell clones and being present on a relatively large number or all T cell clones is not always precise, those skilled in the art to which this invention pertains fully understand and appreciate this demarcation and can readily classify most given antigenic determinants as being either a minor or a major framework determinant. For purposes of this application any antigenic determinant as to which those skilled in the art would not concur is defined to be a minor framework determinant. In general antibodies to major framework determinants will recognize at least 20% of the peripheral T cells of a normal subject and antibodies to minor framework determinants will recognize less than 20% of the peripheral T cells of a normal subject. By contrast, recognition of peripheral T cells of normal subjects by anti-idiotypic antibodies will not be detectable.
Natural Killer (NK) cells, although lacking most of the surface differentiation antigens associated with the three major subsets of T cells, have been confirmed as members of the T cell lineage and are considered by many a subset of T cells. NK cells are characterized by their ability to mediate direct cytotoxicity against specific target cells, e.g. cancer cells, without apparent prior immunization (12 13). It has been observed that some NK cells have on their surfaces a heterodimeric receptor capable of antigen recognition without specific MHC protein associations. For purposes of this application, unless otherwise stated, all references to antigen receptors shall include heterodimeric antigen receptors derived from the surface of T cells and NK cells.
It has been suggested that the T cell antigen receptor is noncovalently associated with the T3 protein complex on the membrane (14). The T3 protein complex is composed of three distinct membrane associated polypeptide chains known as T3-gamma, T3-delta and T3-epsilon (15). This T3 protein-antigen receptor association can be demonstrated by immunoprecipitation studies with antibodies against the T3 protein complex and by comodulation or mutant studies in which the antigen receptor and the T3 protein complex may co-disappear or co-appear on the membrane (16).
Heretofore the measurement of T cell antigen receptors has been limited to the detection of T cell-associated antigen receptors by a variety of immunological techniques involving monoclonal antibodies against the antigen receptor. Such measurements have been routinely accomplished by binding fluorescence conjugated monoclonal antibodies to T cells followed by cellular analysis with a fluorescent-activated cell sorter or similar flow cytometer (9). Such cell analysis provides information about the percent of T cells expressing the receptor in a given sample, however the accuracy and reliability of such routine analysis is limited to 1-2% or more of positive cells in a sample using existing flow cytometers. Furthermore the cell analysis is tedious and often requires fresh, live cells and a skilled, dedicated operator.
The molecular mechanism of T cell antigen receptorantigen interaction is a subject of intensive investigation (13). It has been shown (17) that T cell clones are capable of binding membrane bound antigens associated with MHC proteins. Rao, et al. (28) reported however that some antigens can bind to the antigen reactive T cell clones in the absence of a MHC protein. In a recent report (18) on ligand binding to the T cell antigen receptor, recombinant DNA techniques were used to prepare a hybrid molecule with T cell antigen receptor regions contained within immunoglobulin frameworks. However, no immunological reactivities against the hybrid molecules were described therein.
Between 1975-1981, a series of papers were published on the characterization of "T cell antigen receptors" in mouse and rat systems (19,20,21). These "T cell antigen receptors" are not the "T cell antigen receptor" described herein (see references 1,2,3,6,26). References 19,20, and 21 describe a protein, designated "T cell antigen receptor", with a molecular weight of approximately 150,000-180,000 daltons. This "antigen receptor" is further characterized as a dimer with a protein subunit having a molecular weight of about 70,000 daltons. The chromosomal location of this "antigen receptor's" variable region in mice is on chromosome 12 (21). The T cell antigen receptor described within this application has a molecular weight of about 90,000 daltons (1,2,3). Furthermore, the subunit structures of this antigen receptor consist of three heterodimeric glycosylated polypeptides, designated the alpha, beta, and gamma chains, having molecular weights in mans of approximately 45,000 daltons (2), 40,000 daltons (2), and 30,000 daltons, respectively. The chromosomal locations of the variable regions in mice are: alpha, chromosome 14 (26); beta, chromosome 6 (27); and gamma, chromosome 13 (26).
In 1982, it was reported that the external shedding of the T3 protein by human T cell clones could be artificially induced by adding an anti-T3 protein antibody to these T cells in culture. However, no observation of the spontaneous shedding or release of T3 protein, in vivo or in vitro, was noted (22).
Fujimoto et al. (23) developed an enzyme-linked immunoabsorbent assay (ELISA) for the detection of human T cell surface antigens in soluble form. When sera and culture supernatants from various cell lines were tested, Leu-2 antigen, but not Leu-1 or Leu-3, was found to be present. Applicants are also aware of the pending U.S. patent application, U.S. Ser. No. 724,897, filed Apr. 19, 1985 in the name of David Nelson et al., assigned to the U.S. Government and entitled "Soluble Interleukin-2 Receptor As A Disease Indicator And A Method of Assaying The Same." This patent application, based on the findings of Rubin et al. (24), concerns soluble or released Interleukin-2 Receptor, which is believed to interact with the T cell growth factor Interleukin-2. However, neither the Fujimoto publication, the Nelson patent application, or the Rubin publication discloses or teaches the existence of a released T cell antigen receptor and methods for detecting and measuring it in a biological fluid.
The present invention concerns the discovery of antigen receptors and molecules derived therefrom which are released or present in soluble form in a biological fluid, i.e. they are not bound to a T cell or NK cell. Specific T cell antigen receptors are spontaneously released from the T cells at different metabolic rates under disease conditions than under normal conditions, and become accumulated in body fluids. Rapid, sensitive and inexpensive methods for determining the amount of released antigen receptor or complex thereof in a body fluid are provided. These methods are useful for diagnosing and monitoring diseases in a subject.
Furthermore, a plurality of T cell clones in an individual can simultaneously respond to more than one pathogen (30). Thus it is contemplated that the serum from the individual contains a mixture of distinct types of released T cell antigen receptors. The quantitative measurement of each type of the released receptor allows the diagnosis and monitoring of a patient with multiple infections or a disease involving a plurality of pathogens.
It is well known that both T cells and B cells recognize antigens with specificity (7, 13). Although it is established that T cells, unlike B cells, recognize antigens in association with MHC proteins, it is not clear whether antigenic sites on an antigen which are recognizable by T cells or B cells are related. In a recent report, G.S. Bixler and M.Z. Atassi (25) concluded that the regions of a protein molecule recognized by B cells (antibody binding) may also be recognized by T cells. T cells, however, may recognize additional areas of the molecule to which no detectable antibody responses have thus far been observed. These data suggest that the unique aspect of T cell recognition is useful for detecting disease antigens in diagnostic and therapeutic applications (25). The existence of soluble T cell antigen receptors disclosed herein provides a novel source of reagents for these applications.